We have applied our newly developed radiochemical method for assay of argininosuccinase activity in crude homogenates, to a variety of extrahepatic tissues and to cultured cells derived from two normal human cell lines. The sensitivity and accuracy of the assay is high. It is planned to apply the method to mutant cell lines deficient in argininosuccinase activity and to other tissue and cell sources not previously assayed by a method of comparable sensitivity. It will be possible to establish the lower limit of detection and to discriminate between very low activity and no activity within the very low limit. We plan to develop conditions for a new radiochemical method to assay argininosuccinate synthetase activity, based on the same principles of isotope labeling and product separation by ion exchange chromatography as were incorporated in the radiochemical assay for argininosuccinase activity. The assay will be coupled to argininosuccinase and arginase and will have the convenience that the isotope labeling in the ultimate reaction products will be the same as in the argininosuccinase assay. To advance our studies on the mechanism of action of argininosuccinase, we are resuming studies on the active site of the bovine liver enzyme at the stage of covalent modification with bromo analogues of fumaric acid. Work is contnuing in the direction of preparative scale alkylation of the enzyme for the preparation of peptide fragments after successive stages of enzymatic cleavage in order to isolate small, isotopically labeled, peptides for ultimate identification of the alkylated amino acid within the active site region.